Human Mammary Tumor Cells Research Articles

ChemInform Abstract: HALOGENATED L‐FUCOSE AND D‐GALACTOSE ANALOGS: SYNTHESIS AND METABOLIC EFFECTS

Several new analogues of L-fucose modified in the 2 position and the 5-methyl group have been synthesized as potential plasma-membrane glycoconjugate inhibitors or modifiers, and their biological effects have been studied. 2-Chloro-, 2-bromo-, and 2-iodo-2-deoxy-L-fucose (9a, 9b, and 13, respectively) have been prepared by addition of the appropriate halogen to 3,4-di-O-acetyl-L-fucal, followed by hydrolysis of the anomeric halogen and the acetyl groups. A series of four halogenated 5-methyl analogues of L-fucose (4, X = F, Cl, Br, and I) have been obtained starting from 1,2:3,4-di-O-isopropylidene-L-galactose. The synthesis of this latter compound has been improved. A corresponding series of 6-deoxy-6-halo-D-galactose analogues, which are enantiomers of the 5-(halomethyl)-L-fucose analogues, has also been synthesized. Analogues 4b, 4c, and 9b at 1 x 10(-3) M specifically inhibited the incorporation of L-[3H]fucose into macromolecular components of SW613 human mammary tumor cells. Analogue 13 inhibited the growth of L1210 murine leukemic cells with an IC50 of 6 x 10(5) M in culture. 6-Deoxy-6-fluoro-D-galactose and its enantiomer 4a were found to be effective inhibitors of D-[3H]galactose and L-[3H]fucose incorporation, respectively, into macromolecular components of human mammary tumor cells. The effectiveness of inhibition was reduced with an increase in size of the halogen atom. Analogue 4a and its enantiomer have been tritiated at C-1 and both were found to be activated to a nucleotide sugar, which was followed by incorporation into the macromolecular fraction of SW613 human mammary tumor cells in vitro.

Halogenated L-fucose and D-galactose analogues: synthesis and metabolic effects.

Several new analogues of L-fucose modified in the 2 position and the 5-methyl group have been synthesized as potential plasma-membrane glycoconjugate inhibitors or modifiers, and their biological effects have been studied. 2-Chloro-, 2-bromo-, and 2-iodo-2-deoxy-L-fucose (9a, 9b, and 13, respectively) have been prepared by addition of the appropriate halogen to 3,4-di-O-acetyl-L-fucal, followed by hydrolysis of the anomeric halogen and the acetyl groups. A series of four halogenated 5-methyl analogues of L-fucose (4, X = F, Cl, Br, and I) have been obtained starting from 1,2:3,4-di-O-isopropylidene-L-galactose. The synthesis of this latter compound has been improved. A corresponding series of 6-deoxy-6-halo-D-galactose analogues, which are enantiomers of the 5-(halomethyl)-L-fucose analogues, has also been synthesized. Analogues 4b, 4c, and 9b at 1 x 10(-3) M specifically inhibited the incorporation of L-[3H]fucose into macromolecular components of SW613 human mammary tumor cells. Analogue 13 inhibited the growth of L1210 murine leukemic cells with an IC50 of 6 x 10(5) M in culture. 6-Deoxy-6-fluoro-D-galactose and its enantiomer 4a were found to be effective inhibitors of D-[3H]galactose and L-[3H]fucose incorporation, respectively, into macromolecular components of human mammary tumor cells. The effectiveness of inhibition was reduced with an increase in size of the halogen atom. Analogue 4a and its enantiomer have been tritiated at C-1 and both were found to be activated to a nucleotide sugar, which was followed by incorporation into the macromolecular fraction of SW613 human mammary tumor cells in vitro.

Growth of a human mammary tumour cell line in a serum-free medium.

The MCF-7 line of human mammary carcinoma cells is a well characterised line1 which has retained a number of the properties expressed by breast epithelium in vivo, making it a good model for the in vitro study of normal and neoplastic mammary cells. MCF-7 cells have been reported to possess receptors for oestrogen, androgen, progesterone, glucocorticoid2, insulin3 and L-3, 3′, 5-triiodothyronine (T3)4. These cells also show responses to prolactin5, and dome formation1. This line, like most cells in culture, requires the addition of a serum supplement to the medium for growth. The complex and undefined nature of serum adds a complicating aspect to the design and interpretation of any experiments aimed at understanding the interactions of hormones or drugs with mammary epithelium. Our laboratory has shown that it is possible to replace the serum requirement for a number of cell lines with mixtures of hormones and purified serum factors6–8. We now report that MCF-7 cells may be grown, without a lag or adaptation phase, in a serum-free medium supplemented with physiological levels of insulin, transferrin, epidermal growth factor (EGF), prostaglandin F2α (PGF2α) and cold-insoluble globulin (CIg). This medium will support a growth rate identical to that of cells in medium supplemented with an optimal concentration of fetal calf serum. The further addition of the α-1 serum protein first purified by Holmes9 results in a morphology identical to that of cells in 10% serum. Turkington has previously shown EGF effects on mouse mammary expiant cultures10, and mouse EGF has been reported to be stimulatory for human mammary tumour cells . Insulin and transferrin have been shown to be required for the continuous serum-free growth of the ZR-75-1 human mammary tumour cell line12. We believe that this is the first report of the effects of transferrin, PGF2α, Clg, EGF or the α-1 protein on the MCF-7 line.

Movement of human mammary tumour cells in culture: exclusion of fibroblasts by epithelial territories.

Time-lapse cinematography was used to investigate the movement of confronting populations of human mammary epithelium and stromal cells (fibroblasts). Epithelial cell islands from fibroadenomas and from normal lacteal secretions completely excluded the fibroblasts, and individual cell territories were maintained even in dense cultures. Electron microscopy of the boundary between epithelium and fibroblasts showed that the two cell types made contact. In contrast, epithelial islands from two carcinomas did not retain territorial integrity and allowed penetration of mammary fibroblasts. Confronting homologous eptihelial islands from benign tumours merged, but this was shown to be due to interdigitation rather than free mixing of cell. Epithelial cells moved actively but unlike fibroblasts they retained their neighbour relationships.

Blood group-like activity released by human mammary carcinoma cells in culture.

TUMOUR cells synthesise and release antigenic membrane associated components which circulate either in a free state or complexed with host immunoglobulins1–5. A membrane derived N-like glycoprotein, which inhibited Vicia graminea lectin haemagglutination, has been demonstrated in serum and ascites fluid of mice bearing the Ha subline of TA3 murine mammary adenocarcinoma6. Springer et al.7 have shown that M and N blood group reactive substances were present in both benign and malignant lesions of the human breast. But, T-like Arachis hypogaea reactive substances were only found in malignant breast tissue and not in benign tumours or healthy breast tissue7. Isolation and long term cultivation of a human mammary tumour cell line. BOT-2 was recently reported8. Immunological tests indicated that women with diagnosed mammary cancer had circulating antibodies that reacted with these cells9 and induced cell surface shedding10. Here we report the use of blood-typing antisera and lectins to characterise the proteins synthesised and spontaneously released from BOT-2 cells growing in defined medium. To our knowledge this is the first demonstration of M, N, T, Tn erythrocyte-like glycoproteins being spontaneously shed from human epithelial tumour cells in culture.

Problems of In Vitro Culture of Human Mammary Tumor Cells

Various techniques were used to obtain cell cultures from benign and malignant tumors removed from human breasts. Cell suspensions were prepared by trypsinizing finely minced tissue or by the spilling technique or Lasfargues and Ozzello, and T-flasks or T-flasks coated with collagen were inoculated. Trypsinized tissue yielded predominantly fibroblastic cultures, whereas those obtained by the spilling technique were rich in epithelial cells. The mixed cell populations were separated into pure suspensions by density gradient centrifugation. Most epithelial cell cultures grew best when they were collected by the spilling technique and inoculated into collagen-coated T-flasks with Eagle's medium enriched with 30% fetal calf serum. However, tears developed in the collagen coat with subsequent peeling which led to the loss of many epithelial cell colonies. Primary epithelial cells did not remain viable for prolonged periods, and attempts to obtain subcultures were unsuccessful. In this Paper the work of several investigators is reviewed and their methods of growing breast tumor cell are discussed.